[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR799.9430]
[Page 407-416]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE
TESTING REQUIREMENTS--Table of Contents
Subpart H_Health Effects Test Guidelines
Sec. 799.9430 TSCA combined chronic toxicity/carcinogenicity.
(a) Scope. This section is intended to meet the testing requirements
under section 4 of the Toxic Substances Control Act (TSCA). The
objective of a combined chronic toxicity/carcinogenicity study is to
determine the effects of a substance in a mammalian species following
prolonged and repeated exposure. The application of this section should
generate data which identify the majority of chronic and carcinogenicity
effects and determine dose-response relationships. The design and
conduct should allow for the detection of neoplastic effects and a
determination of the carcinogenic potential as well as general toxicity,
including neurological, physiological, biochemical, and hematological
effects and exposure-related morphological (pathology) effects.
(b) Source. The source material used in developing this TSCA test
guideline is the Office of Prevention, Pesticides, and Toxic Substances
(OPPTS) harmonized test guideline 870.4300 (August 1998, final
guideline). This source is available at the address in paragraph (h) of
this section.
(c) Definitions. The following definitions apply to this section.
Carcinogenicity is the development of neoplastic lesions as a result
of the repeated daily exposure of experimental animals to a chemical by
the oral, dermal, or inhalation routes of exposure.
Chronic toxicity is the adverse effects occurring as a result of the
repeated daily exposure of experimental animals to a chemical by the
oral, dermal, or inhalation routes of exposure.
Cumulative toxicity is the adverse effects of repeated dose
occurring as a result of prolonged action on, or increased concentration
of, the administered test substance or its metabolites in susceptible
tissues.
Dose in a combined chronic toxicity/carcinogenicity study is the
amount of test substance administered via the oral, dermal, or
inhalation routes for a period of up to 24 months. Dose is expressed as
weight of the test substance per unit body weight of test animal
(milligrams per kilogram), or as weight of the test substance in parts
per million (ppm) in food or drinking water. When exposed via
inhalation, dose is expressed as weight of the test substance per unit
volume of air (milligrams per liter) or as parts per million per day.
For dermal application, dose is expressed as weight of the test
substance (grams, milligrams) per unit
[[Page 408]]
body weight of the test animal (milligrams per kilogram) or as weight of
the substance per unit surface area (milligrams per square centimeter)
per day.
No-observed-effects level (NOEL) is the maximum dose used in a study
which produces no observed adverse effects. The NOEL is usually
expressed in terms of the weight of a test substance given daily per
unit weight of test animal (milligrams per kilogram per day).
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg
body weight (expected human exposure may indicate the need for a higher
dose level), using the procedures described for this study, produces no
observable toxic effects or if toxic effects would not be expected based
upon data of structurally related compounds, then a full study using
three dose levels might not be necessary.
(e) Test procedures--(1) Animal selection--(i) Species and strain.
Preliminary studies providing data on acute, subchronic, and metabolic
responses should have been carried out to permit an appropriate choice
of animals (species and strain). As discussed in other guidelines, the
mouse and rat have been most widely used for assessment of carcinogenic
potential, while the rat and dog have been most often studied for
chronic toxicity. For the combined chronic toxicity/carcinogenicity
study via the oral and inhalation routes, the rat is the species of
choice and for the dermal route, the mouse is species of choice. If
other species are used, the tester must provide justification/reasoning
for their selection. The strain selected should be susceptible to the
carcinogenic or toxic effect of the class of substances being tested, if
known, and provided it does not have a spontaneous background incidence
too high for meaningful assessment. Commonly used laboratory strains
must be employed.
(ii) Age/weight. (A) Testing must be started with young healthy
animals as soon as possible after weaning and acclimatization.
(B) Dosing should generally begin no later than 8 weeks of age.
(C) At commencement of the study, the weight variation of animals
used must be within 20% of the mean weight for each sex.
(D) Studies using prenatal or neonatal animals may be recommended
under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex must be used at
each dose level.
(B) Females must be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 100 rodents (50 males and 50 females)
must be used at each dose level and concurrent control group. At least
20 additional rodents (10 males and 10 females) should be used for
satellite dose groups and the satellite control group. The purpose of
the satellite group is to allow for the evaluation of chronic toxicity
after 12 months of exposure to the test substance.
(B) For a meaningful and valid statistical evaluation of long term
exposure and for a valid interpretation of negative results, the number
of animals in any group should not fall below 50% at 15 months in mice
and 18 months in rats. Survival in any group should not fall below 25%
at 18 months in mice and 24 months in rats.
(C) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required.
(D) Each animal must be assigned a unique identification number.
Dead animals (and their preserved organs) and tissues, and microscopic
slides shall be identified by reference to the unique numbers assigned.
(v) Husbandry. (A) Animals may be group-caged by sex, but the number
of animals per cage must not interfere with clear observation of each
animal. The biological properties of the test substance or toxic effects
(e.g., morbidity, excitability) may indicate a need for individual
caging. Rodents should be housed individually in dermal studies and
during exposure in inhalation studies.
(B) The temperature of the experimental animal rooms should be at 22
3 [deg]C.
(C) The relative humidity of the experimental animal rooms should be
50 20%.
[[Page 409]]
(D) Where lighting is artificial, the sequence should be 12 hours
light/12 hours dark.
(E) Control and test animals should be fed from the same batch and
lot. The feed should be analyzed to assure uniform distribution and
adequacy of nutritional requirements of the species tested and for
impurities that might influence the outcome of the test. Animals should
be fed and watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until animals have been
allowed a period of acclimatization/quarantine to environmental
conditions, nor should animals from outside sources be placed on test
without an adequate period of quarantine. An acclimation period of at
least five days is recommended.
(2) Control and test substances. (i) Where necessary, the test
substance is dissolved or suspended in a suitable vehicle. If a vehicle
or diluent is needed, it should not elicit toxic effects itself nor
substantially alter the chemical or toxicological properties of the test
substance. It is recommended that wherever possible the usage of an
aqueous solution be considered first, followed by consideration of a
solution in oil, and finally solution in other vehicles.
(ii) One lot of the test substance should be used throughout the
duration of the study if possible, and the research sample should be
stored under conditions that maintain its purity and stability. Prior to
the initiation of the study, there should be a characterization of the
test substance, including the purity of the test compound, and, if
possible, the name and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into
feed or another vehicle, the period during which the test substance is
stable in such a mixture should be determined prior to the initiation of
the study. Its homogeneity and concentration should be determined prior
to the initiation of the study and periodically during the study.
Statistically randomized samples of the mixture should be analyzed to
ensure that proper mixing, formulation, and storage procedures are being
followed, and that the appropriate concentration of the test or control
substance is contained in the mixture.
(3) Control groups. A concurrent control group is required. This
group should be an untreated or sham-treated control group or, if a
vehicle is used in administering the test substance, a vehicle control
group. If the toxic properties of the vehicle are not known or cannot be
made available, both untreated and vehicle control groups are required.
(4) Dose levels and dose selection. (i) For risk assessment
purposes, at least three dose levels must be used, in addition to the
concurrent control group. Dose levels should be spaced to produce a
gradation of effects. A rationale for the doses selected must be
provided.
(ii) The highest dose level in rodents should elicit signs of
toxicity without substantially altering the normal life span due to
effects other than tumors. The highest dose should be determined based
on the findings from a 90-day study to ensure that the dose used is
adequate to assess the chronic toxicity and the carcinogenic potential
of the test substance. Thus, the selection of the highest dose to be
tested is dependent upon changes observed in several toxicological
parameters in subchronic studies. The highest dose tested need not
exceed 1,000 mg/kg/day.
(iii) The intermediate-dose levels should be spaced to produce a
gradation of toxic effects.
(iv) The lowest-dose level should produce no evidence of toxicity.
(v) For skin carcinogenicity studies, when toxicity to the skin is a
determining factor, the highest dose selected should not destroy the
functional integrity of the skin, the intermediate doses should be a
minimally irritating dose and the low dose should be the highest
nonirritating dose.
(vi) The criteria for selecting the dose levels for skin
carcinogenicity studies, based on gross and histopathologic dermal
lesions, are as follows:
(A) Gross criteria for reaching the high dose:
(1) Erythema (moderate).
(2) Scaling.
(3) Edema (mild).
(4) Alopecia.
(5) Thickening.
[[Page 410]]
(B) Histologic criteria for reaching the high dose:
(1) Epidermal hyperplasia.
(2) Epidermal hyperkeratosis.
(3) Epidermal parakeratosis.
(4) Adnexal atrophy/hyperplasia.
(5) Fibrosis.
(6) Spongiosis (minimal-mild).
(7) Epidermal edema (minimal-mild).
(8) Dermal edema (minimal-moderate).
(9) Inflammation (moderate).
(C) Gross criteria for exceeding the high dose:
(1) Ulcers-fissures, exudate/crust (eschar), nonviable (dead)
tissues.
(2) Anything leading to destruction of the functional integrity of
the epidermis (e.g., caking, fissuring, open sores, eschar).
(D) Histologic criteria for exceeding the high-dose:
(1) Crust (interfollicular and follicular).
(2) Microulcer.
(3) Degeneration/necrosis (mild to moderate).
(4) Epidermal edema (moderate to marked).
(5) Dermal edema (marked).
(6) Inflammation (marked).
(5) Administration of the test substance. The three main routes of
administration are oral, dermal, and inhalation. The choice of the route
of administration depends upon the physical and chemical characteristics
of the test substance and the form typifying exposure in humans.
(i) Oral studies. If the test substance is administered by gavage,
the animals are dosed with the test substance on a 7-day per week basis
for a period of at least 18 months for mice and hamsters and 24 months
for rats. However, based primarily on practical considerations, dosing
by gavage on a 5-day per week basis is acceptable. If the test substance
is administered in the drinking water or mixed in the diet, then
exposure should be on a 7-day per week basis.
(ii) Dermal studies. (A) Preparation of animal skin. Shortly before
testing, fur should be clipped from not less than 10% of the body
surface area for application of the test substance. In order to dose
approximately 10% of the body surface, the area starting at the scapulae
(shoulders) to the wing of the ileum (hipbone) and half way down the
flank on each side of the animal should be shaved. Shaving should be
carried out approximately 24 hours before dosing. Repeated clipping or
shaving is usually needed at approximately weekly intervals. When
clipping or shaving the fur, care should be taken to avoid abrading the
skin which could alter its permeability.
(B) Preparation of test substance. Liquid test substances are
generally used undiluted, except as indicated in paragraph (e)(4)(vi) of
this section. Solids should be pulverized when possible. The substance
should be moistened sufficiently with water or, when necessary, with a
suitable vehicle to ensure good contact with the skin. When a vehicle is
used, the influence of the vehicle on toxicity of, and penetration of
the skin by, the test substance should be taken into account. The volume
of application should be kept constant, e.g., less than 100 [micro]L for
the mouse and less than 300 [micro]L for the rat. Different
concentrations of test solution should be prepared for different dose
levels.
(C) Administration of test substance. The duration of exposure
should be at least 18 months for mice and hamsters and 24 months for
rats. Ideally, the animals should be treated with test substance for at
least 6 hours per day on a 7-day per week basis. However, based on
practical considerations, application on a 5-day per week basis is
acceptable. Dosing should be conducted at approximately the same time
each day. The test substance must be applied uniformly over the
treatment site. The surface area covered may be less for highly toxic
substances. As much of the area should be covered with as thin and
uniform a film as possible. For rats, the test substance may be held in
contact with the skin with a porous gauze dressing and nonirritating
tape if necessary. The test site should be further covered in a suitable
manner to retain the gauze dressing plus test substance and to ensure
that the animals cannot ingest the test substance. The application site
should not be covered when the mouse is the species of choice. The test
substance may
[[Page 411]]
be wiped from the skin after the 6-hour exposure period to prevent
ingestion.
(iii) Inhalation studies. (A) The animals should be exposed to the
test substance, for 6 hours per day on a 7-day per week basis, for a
period of at least 18 months in mice and 24 months in rats. However,
based primarily on practical considerations, exposure for 6 hours per
day on a 5-day per week basis is acceptable.
(B) The animals must be tested in dynamic inhalation equipment
designed to sustain a minimum air flow of 10 air changes per hour, an
adequate oxygen content of at least 19%, and uniform conditions
throughout the exposure chamber. Maintenance of slight negative pressure
inside the chamber will prevent leakage of the test substance into
surrounding areas. It is not normally necessary to measure chamber
oxygen concentration if airflow is adequate.
(C) The selection of a dynamic inhalation chamber should be
appropriate for the test substance and test system. Where a whole body
chamber is used, individual housing must be used to minimize crowding of
the test animals and maximize their exposure to the test substance. To
ensure stability of a chamber atmosphere, the total volume occupied by
the test animals shall not exceed 5% of the volume of the test chamber.
It is recommended, but not required, that nose-only or head-only
exposure be used for aerosol studies in order to minimize oral exposures
due to animals licking compound off their fur. The animals should be
acclimated and heat stress minimized.
(D) The temperature at which the test is performed should be
maintained at 22 2 [deg]C. The relative humidity
should be maintained between 40 to 60%, but in certain instances (e.g.,
tests of aerosols, use of water vehicle) this may not be practicable.
(E) The rate of air flow must be monitored continuously but recorded
at least three times during the exposure.
(F) Temperature and humidity must be monitored continuously but
should be recorded at least every 30 minutes.
(G) The actual concentrations of the test substance must be measured
in the animal's breathing zone. During the exposure period, the actual
concentrations of the test substance must be held as constant as
practicable and monitored continuously or intermittently depending on
the method of analysis. Chamber concentration may be measured using
gravimetric or analytical methods as appropriate. If trial run
measurements are reasonably consistent (10% for
liquid aerosol, gas, or vapor; 20% for dry
aerosol), then two measurements should be sufficient. If measurements
are not consistent, three to four measurements should be taken. If there
is some difficulty in measuring chamber analytical concentration due to
precipitation, nonhomogeneous mixtures, volatile components, or other
factors, additional analyses of inert components may be necessary.
(H) During the development of the generating system, particle size
analysis must be performed to establish the stability of aerosol
concentrations with respect to particle size. The mass median
aerodynamic diameter (MMAD) particle size range should be between 1-3
[micro]m. The particle size of hygroscopic materials should be small
enough when dry to assure that the size of the swollen particle will
still be within the 1-3 [micro]m range. Measurements of aerodynamic
particle size in the animal's breathing zone should be measured during a
trial run. If MMAD values for each exposure level are within 10% of each
other, then two measurements during the exposures should be sufficient.
If pretest measurements are not within 10% of each other, three to four
measurements should be taken.
(I) Feed must be withheld during exposure. Water may also be
withheld during exposure.
(J) When the physical and chemical properties of the test substance
show a low flash point or the test substance is otherwise known or
thought to be explosive, care must be taken to avoid exposure level
concentrations that could result in an exposure chamber explosion during
the test.
(6) Observation period. (i) This time period must not be less than
24 months for rats and 18 months for mice, and ordinarily not longer
than 30 months for rats and 24 months for mice. For longer time periods,
and where any other species are used, consultation with the
[[Page 412]]
Agency in regard to the duration of the study is advised.
(ii) Animals in a satellite group to assess chronic toxicity should
be observed for 12 months.
(7) Observation of animals. (i) Observations must be made at least
twice each day for morbidity and mortality. Appropriate actions should
be taken to minimize loss of animals to the study (e.g., necropsy or
refrigeration of those animals found dead and isolation or sacrifice of
weak or moribund animals). General clinical observations shall be made
at least once a day, preferably at the same time each day, taking into
consideration the peak period of anticipated effects after dosing. The
clinical condition of the animal should be recorded.
(ii) A careful clinical examination must be made at least once
weekly. Observations should be detailed and carefully recorded,
preferably using explicity defined scales. Observations should include,
but not be limited to, evaluation of skin and fur, eyes and mucous
membranes, respiratory and circulatory effects, autonomic effects such
as salivation, central nervous system effects, including tremors and
convulsions, changes in the level of activity, gait and posture,
reactivity to handling or sensory stimuli, altered strength, and
stereotypes or bizarre behavior (e.g., self-mutilation, walking
backwards).
(iii) Signs of toxicity should be recorded as they are observed
including the time of onset, degree and duration.
(iv) Body weights must be recorded individually for all animals once
prior to administration of the test substance, once a week during the
first 13 weeks of the study and at least once every 4 weeks thereafter
unless signs of clinical toxicity suggest more frequent weighing to
facilitate monitoring of health status.
(v) Measurements of feed consumption should be determined weekly
during the first 13 weeks of the study and then at approximately monthly
intervals unless health status or body weight changes dictate otherwise.
Measurements of water consumption should be determined at the same
intervals if the test material is administered in drinking water.
(vi) Moribund animals must be removed and sacrificed when noticed
and the time of death should be recorded as precisely as possible. At
the end of the study period, all survivors must be sacrificed. Animals
in the satellite group must be sacrificed after 12 months of exposure to
the test substance (interim sacrifice).
(8) Clinical pathology. Hematology, clinical chemistry and
urinalyses must be performed from 10 animals per sex per group. The
parameters should be examined at approximately 6 month intervals during
the first 12 months of the study. If possible, these collections should
be from the same animals at each interval. If hematological and
biochemical effects are seen in the subchronic study, testing shall also
be performed at 3 months. Overnight fasting of animals prior to blood
sampling is recommended.
(i) Hematology. The recommended parameters are red blood cell count,
hemoglobin concentration, hematocrit, mean corpuscular volume, mean
corpuscular hemoglobin, and mean corpuscular hemoglobin concentration,
white blood cell count, differential leukocyte count, platelet count,
and a measure of clotting potential, such as prothrombin time or
activated partial thromboplastin time.
(ii) Clinical chemistry. (A) Parameters which are considered
appropriate to all studies are electrolyte balance, carbohydrate
metabolism, and liver and kidney function. The selection of specific
tests will be influenced by observations on the mode of action of the
substance and signs of clinical toxicity.
(B) The recommended clinical chemistry determinations are potassium,
sodium, glucose, total cholesterol, urea nitrogen, creatinine, total
protein, and albumin. More than two hepatic enzymes, (such as alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase,
sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be
measured. Measurements of addtional enzymes (of hepatic or other origin)
and bile acids, may also be useful.
(iii) If a test chemical has an effect on the hematopoietic system,
[[Page 413]]
reticulocyte counts and bone marrow cytology may be indicated.
(iv) Other determinations that should be carried out if the test
chemical is known or suspected of affecting related measures include
calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and
cholinesterases.
(v) Urinalyses. Urinalysis for rodents must be performed at the end
of the first year of the study using timed urine collection. Urinalysis
determinations include: appearance, volume, osmolality or specific
gravity, pH, protein, glucose, and blood/blood cells.
(9) Ophthalmological examination. Examinations must be made on all
animals using an ophthalmoscope or an equivalent device prior to the
administration of the test substance and at termination of the study on
10 animals per sex in the high-dose and control groups. If changes in
eyes are detected, all animals must be examined.
(10) Gross necropsy. (i) A complete gross examination must be
performed on all animals, including those which died during the
experiment or were sacrificed in a moribund condition.
(ii) At least, the liver, kidneys, adrenals, testes, epididymides,
ovaries, uterus, spleen, brain, and heart should be trimmed and weighed
wet, as soon as possible after dissection to avoid drying. The lungs
should be weighed if the test substance is administered by the
inhalation route. The organs should be weighed from interim sacrifice
animals as well as from at least 10 animals per sex per group at
terminal sacrifice.
(iii) The following organs and tissues, or representative samples
thereof, must be preserved in a suitable medium for possible future
histopathological examination:
(A) Digestive system--salivary glands, esophagus, stomach, duodenum,
jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when
present) .
(B) Nervous system--brain (multiple sections, including cerebrum,
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or
tibial, preferably in close proximity to the muscle), spinal cord (three
levels, cervical, mid-thoracic, and lumbar), eyes (retina, optic nerve).
(C) Glandular system--adrenals, parathyroid, thyroid.
(D) Respiratory system--trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/Hematopoietic system--aorta, heart, bone marrow
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering
the route of administration and another one distant from the route of
administration to cover systemic effects), spleen.
(F) Urogenital system--kidneys, urinary bladder, prostate, testes,
epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Other--all gross lesions and masses, skin.
(iv) In inhalation studies, the entire respiratory tract, including
nose, pharynx, larynx, and paranasal sinuses should be examined and
preserved. In dermal studies, skin from treated and adjacent control
skin sites should be examined and preserved.
(v) Inflation of lungs and urinary bladder with a fixative is the
optimal method for preservation of these tissues. The proper inflation
and fixation of the lungs in inhalation studies is essential for
appropriate and valid histopathological examination.
(vi) Information from clinical pathology and other in-life data
should be considered before microscopic examination, since these data
may provide significant guidance to the pathologist.
(11) [Reserved]
(12) Histopathology. (i) The following histopathology must be
performed:
(A) Full histopathology on the organs and tissues, listed in
paragraph (e)(10)(iii) of this section of all animals in the control and
high dose groups and of all animals that died or were sacrificed during
the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) If the results show substantial alteration of the animal's
normal life span, the induction of effects that might affect a
neoplastic response, or other effects that might compromise the
significance of the data, the next lower levels should be examined fully
as described in paragraph (e)(12)(i) of this section.
[[Page 414]]
(iii) An attempt should be made to correlate gross observations with
microscopic findings.
(iv) Tissues and organs designated for microscopic examination
should be fixed in 10% buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours
prior to trimming.
(f) Data and reporting--(1) Treatment of results. (i) Data must be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions and the percentage of animals displaying each type
of lesion.
(ii) When applicable, all observed results, quantitative and
qualitative, must be evaluated by an appropriate statistical method. Any
generally accepted statistical methods may be used; the statistical
methods including significance criteria should be selected during the
design of the study.
(2) Evaluation of study results. (i) The findings of a combined
chronic toxicity/carcinogenicity study should be evaluated in
conjunction with the findings of previous studies and considered in
terms of the toxic effects, the necropsy and histopathological findings.
The evaluation must include the relationship between the dose of the
test substance and the presence, incidence and severity of abnormalities
(including behavioral and clinical abnormalities), gross lesions,
identified target organs, body weight changes, effects on mortality and
any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailablity of the
test substance should be considered.
(iii) In order for a negative test to be acceptable, it should meet
the following criteria--no more than 10% of any group is lost due to
autolysis, cannibalism, or management problems, and survival in each
group is no less than 50% at 15 months for mice and 18 months for rats.
Survival should not fall below 25% at 18 months for mice and 24 months
for rats.
(iv) The use of historical control data from an appropriate time
period from the same testing laboratory (i.e, the incidence of tumors
and other suspect lesions normally occurring under the same laboratory
conditions and in the same strain of animals employed in the test) is
helpful for assessing the significance of changes observed in the
current study.
(3) Test report. (i) In addition to the reporting requirements
specified under EPA Good Laboratory Practice Standards at 40 CFR part
792, subpart J, the following specific information must be reported:
(A) Test substance characterization should include:
(1) Chemical identification.
(2) Lot or batch number.
(3) Physical properties.
(4) Purity/impurities.
(5) Identification and composition of any vehicle used.
(B) Test system should contain data on:
(1) Species and strain of animals used and rationale for selection
if other than that recommended.
(2) Age including body weight data and sex.
(3) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods.
(4) Identification of animal diet.
(5) Acclimation period.
(C) Test procedure should include the following data:
(1) Method of randomization used.
(2) Full description of experimental design and procedure.
(3) Dose regimen including levels, methods, and volume.
(4) Test results. (i) Group animal data. Tabulation of toxic
response data by species, strain, sex, and exposure level for:
(A) Number of animals exposed.
(B) Number of animals showing signs of toxicity.
(C) Number of animals dying.
(ii) Individual animal data. Data should be presented as summary
(group mean) as well as for individual animals.
(A) Time of death during the study or whether animals survived to
termination.
(B) Time of observation of each abnormal sign and its subsequent
course.
(C) Body weight data.
[[Page 415]]
(D) Feed and water consumption data, when collected.
(E) Achieved dose (milligrams/kilogram body weight) as a time-
weighed average is the test substance is administered in the diet or
drinking water.
(F) Results of ophthalmological examination, when performed.
(G) Results of hematological tests performed.
(H) Results of clinical chemistry tests performed.
(I) Results of urinalysis tests performed.
(J) Results of observations made.
(K) Necropsy findings including absolute/relative organ weight data.
(L) Detailed description of all histopathological findings.
(M) Statistical treatment of results where appropriate.
(N) Historical control data.
(iii) In addition, for inhalation studies the following should be
reported:
(A) Test conditions. The following exposure conditions must be
reported.
(1) Description of exposure apparatus including design, type,
dimensions, source of air, system for generating particulates and
aerosols, method of conditioning air, treatment of exhaust air and the
method of housing the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size should be described.
(B) Exposure data. These must be tabulated and presented with mean
values and a measure of variability (e.g., standard deviation) and
should include:
(1) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Actual (analytical or gravimetric) concentration in the
breathing zone.
(4) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(5) Particle size distribution, and calculated MMAD and geometric
standard deviation.
(6) Explanation as to why the desired chamber concentration and/or
particle size could not be achieved (if applicable) and the efforts
taken to comply with this aspect of the guidelines.
(g) Quality control. A system must be developed and maintained to
assure and document adequate performance of laboratory equipment. The
study must be conducted in compliance with 40 CFR Part 792--Good
Laborary Practice Standards.
(h) References. For additional background information on this test
guideline, the following references should be consulted. These
references are available for inspection at the TSCA Nonconfidential
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,
except legal holidays.
(1) Benitz, K.F. Measurement of Chronic Toxicity. Methods of
Toxicology. Ed. G.E. Paget. Blackwell, Oxford. pp. 82-131 (1970).
(2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W.,
Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity
Experiments: Implication for Neurotoxicological Assessments.
Neurotoxicol. Teratol. 13, 599-609. (1991)
(3) D'Aguanno, W. Drug Safety Evaluation--Pre-Clinical
Considerations. Industrial Pharmacology: Neuroleptic. Vol. I, Ed. S.
Fielding and H. Lal. Futura, Mt. Kisco, NY. pp. 317-332 (1974).
(4) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and
Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing
1975).
(5) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs,
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The
Association of Food and Drug Officials of the United States. pp. 60-67
(1959, 3rd Printing 1975).
(6) Organization for Economic Cooperation and Development.
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 453
Combined Chronic Toxicity/Carcinogenicity Studies, Paris. (1981).
(7) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines.
Journal of Environmental Pathology and Toxicology 11:161-182 (1977).
(8) Page, N.P. Concepts of a Bioassay Program in Environmental
Carcinogenesis, Advances in Modern Toxicology. Vol.3, Ed. Kraybill and
[[Page 416]]
Mehlman. Hemisphere, Washington, DC pp. 87-171 (1977)
(9) Sontag, J.M. et al. Guidelines for Carcinogen Bioassay in Small
Rodents. NCI-CS-TR-1 (Bethesda: United States Cancer Institute, Division
of Cancer Control and Prevention, Carcinogenesis Bioassay Program.
(10) Summary of the EPA Workshop on Carcinogenesis Bioassay via the
Dermal Route. EPA Report 50/6-89-002; 50/6-89-003. Washington, DC.
(11) The Atlas Of Dermal Lesions, EPA Report 20T-004, U.S
Environmental Protection Agency, Washington, DC.
[65 FR 78802, Dec. 15, 2000]